Glycoprotein is a factor involved in various life activities of the human body. Thus, it is important to understand the functional changes of the glycoprotein. Proteins produced in cells are translated according to their functions and then modified, which is ‘post-translational modification’, by which they become different glycoproteins having diverse sugar chain structures and accordingly having different unique functions. Glycosylation is the most representative protein modification. Secretory proteins derived from cancer cells or surface proteins of cell membrane are non-specifically glycosylated by oncogene, thereby causing cancer. The relationship between cancer development and glycosylation has been studied extensively and the results confirmed that cancer could be developed by the cancer-specific glycosylation (Salome S. et al., Nature Reviews Cancer, 2015, 15(9):540-550).
The abnormal glycosylation in glycoprotein is fucosylation, sialylation, and polylactosamine, etc. Analysis of such abnormal glycosylation can be used to collect clinical information for cancer diagnosis and prognosis. Such glycoproteins that can give clinically useful information can be used as cancer biomarkers. Effective methods for the development and validation of cancer biomarkers have been developed. A glycoprotein capable of providing cancer-related information can be used to diagnose cancer by detecting the glycoprotein in a culture medium of cancer cells, a lysate of cancer tissue, or blood. At this time, blood which is easy to collect as a sample is mainly used. Therefore, it is important to analyze glycoprotein, the cancer biomarker, in a blood sample. However, the amount of glycoprotein in blood is very small for efficient analysis, so an effective method with high sensitivity and accuracy is required.
To diagnose cancer, a technique that can clearly distinguish cancer patients from normal people is important. In particular, various analytical methods have been developed to analyze protein glycosylation for cancer diagnosis. Among those methods, an analytical method for separating and concentrating a target sugar chain to be analyzed using a lectin protein that reacts with a specific sugar chain is most widely used. The blood used as an analytical sample has a rather complicated composition, so the method to separate and concentrate glycoproteins using a lectin protein seems more efficient for the analysis of a specific sugar chain structure. The separated and concentrated glycoprotein can be analyzed by spectroscopic methods to obtain the final results.
Lectin-blotting is an example of the glycoprotein analysis method using a lectin protein. This method is based on immunoblotting using a specific antibody selectively binding to the glycoprotein having a specific sugar chain structure. The lectin-blotting method can selectively separate and concentrate the marker glycoprotein having a specific sugar chain structure by using the selective binding capacity of lectin to the sugar chain structure of the glycoprotein. In the method above, various lectin proteins such as ConA (concanavalin A), WGA (wheat germ agglutinin), jacalin, SNA (sambucus nigra agglutinin), AAL (aleuria aurantia lectin), LPHA (phytohemagglutinin-L), PNA (peanut agglutinin), LCA (lens culimaris agglutinin-A), and DSA (datura stramonium agglutinin) can be used according to the structure of the sugar chain to be separated and concentrated (Yang Z. et al., J. Chromatography, 2001, 1053:79-88; Wang Y. et al., Glycobiology, 2006, 16:514-523). However, the lectin-blotting method is a gel-based analysis technique, so the analysis speed and the reliability of the result might be limited.
The unsatisfactory analysis speed and reliability can be improved by using lectin/enzyme-linked immunosorbent assay (lectin/ELISA) based on sandwich array (Forrester S. et al., Cancer Mol. Oncol., 2007, 1(2):216-225). Sandwich array uses the primary and the secondary antibodies for the analysis. At this time, non-specific reactions can be occurred due to the cross-reactivity of the secondary antibodies, which might results in poor reproducibility of results. This method is not so economical because of using two antibodies.
In the meantime, high-speed/high-sensitivity qualitative/quantitative analysis using a mass spectrometer is also used for the analysis of glycoprotein. In particular, multiple reaction monitoring mass spectrometry (MRM) facilitates quantification of the peptide produced from protein hydrolysis, which is highly reliable. This method allows relatively quick and accurate analysis results from the blood samples having complicated composition. According to MRM, a target peptide generated from the hydrolysis of a target glycoprotein is analyzed by liquid chromatography at least once and precursor mass selection and product ion selection at least twice, enabling selective analysis with high sensitivity using the sample as blood.
Recently, a parallel reaction monitoring (PRM) technique has been known (Peterson et al., Mol. Cell Proteomics, 2012, 11(11):1475-1488). Unlike MRM, this method uses a mass spectrometer equipped with a trap and time-of-flight mass analyzer, so that a product ion spectrum of the peptide can be obtained, allowing quantitative and qualitative analysis of the peptide simultaneously. This method can analyze trace glycoproteins that exhibit low signals with high reproducibility and excellent sensitivity (Kim et al., Analytica Chimica acta., 2015(882):38-48).
The methods for analyzing a specific sugar chain using a mass spectrometer include the method based on the analysis of the sugar chain separated from glycoproteins, the method based on the analysis of the sugar chain bound glycopeptide, and the method based on the analysis of the sugar chain bound glycoprotein. The sugar chains bound to the protein by modification have various structures and exhibit heterogeneity of the sugar chain having various structures at the same amino acid position and at the same time. It is also expected that the position of the amino acid to which the sugar chain can bind varies, and the role of the sugar chain varies depending on the position. Therefore, it is important to analyze the glycopeptide site-specifically.
Proteins are rich in serum that has been used as a sample for disease diagnosis. Among them, about 10 high concentration proteins take almost 90% of the total mass of blood. However, well known biomarker proteins have a relatively low concentration, which makes the accurate detection in a sample difficult (Anderson N. L. et al., Cell Proteomics, 2002, 1:845-867). Therefore, a pretreatment process that minimizes the complexity of the serum is required to analyze the biomarkers in the serum. To do so, such methods as depletion to eliminate the high concentration proteins and antibody-based immunoprecipitation to selectively concentrate a target protein can be used. In particular, when a selected biomarker is well known as in the case of cancer, it is efficient to use an immunoprecipitation method by selecting an effective antibody against the biomarker.
Hepatocellular carcinoma is about 76% of total liver cancer. The causes of hepatocellular carcinoma include chronic hepatitis B, chronic hepatitis C, cirrhosis, alcoholic liver disease, and diabetic liver disease. In particular, hepatitis B is a high risk factor, which is the cause of 72% of the total hepatocellular carcinoma in Korea. Hepatocellular carcinoma occurs in about one third of patients with liver cirrhosis. Therefore, it is highly desirable to develop a cancer biomarker capable of diagnosing progression from liver disease to hepatocellular carcinoma. Hepatocellular carcinoma does not show symptoms until it progresses significantly. So, a regular examination is needed, otherwise the treatment is very limited. In order for the treatment to be efficient, early diagnosis technique is required.
Therefore, in the course of study to identify a biomarker useful for the diagnosis of cancer, the present inventors confirmed that liver cancer patients and liver disease patients such as cirrhosis patients or hepatitis patients can be distinguished by calculating the rate of fucosylation of AFP glycopeptide, leading to the completion of the present invention.